Because soybeans are a good source of oil, isoflavones and proteins, genetic engineering of soybeans for higher value cultivars has tremendous market value. Soybean transformation procedures include gene gun bombardment of somatic embryos. The bombarded embryos are selected with a selectable marker that is part of the introduced DNA. For soybean somatic embryo selection, the antibiotic hygromycin is often used. Market forces and health and safety concerns have created a need to eliminate such genes from improved crop varieties, but existing processes are time consuming. There is a need for non-antibiotic selection maker genes that can eliminate these problems. Only a limited number of non-antibiotic resistance markers are available for plant improvement. For example, herbicide selection systems like glyphosate may be useful but are generally not available for use.
Soybean cellular selection systems are exceptional in that they are recalcitrant to selection that commonly works with many plant cellular selection systems. For example, the most common cellular selection system for plant systems is kanamycin resistance conferred by an nptII gene, but after numerous failed attempts it was concluded that this selection system cannot be accomplished with soybeans. Antibiotic resistance markers have been used in seeds to produce transgenic canola, tobacco, barley, and soybean plants that express a bacterial variant of DHPS that is insensitive to AEC. The presence of antibiotic resistance genes in these plants is an undesirable result.
AEC is a lysine analog that naturally occurs in the mushroom Rozites caperta (Matsumoto, 1984; Cadogan et al., 1996). It also can be synthesized using N-(tert-butoxycarbonyl)serine and ethanolamine (Arnold et al., 1988). AEC is an inhibitor of dihydrodipicolinate synthase (DHPS or DHDPS) killing cells and tissues due to an inability to synthesize lysine (Perl et al., 1993; Ghislain et al., 1995; Vauterin et al., 2000). AEC also inhibits AK and lysine is another inhibitor of DHPS (Negrutiu et al., 1984). It is known that the DHPS enzyme of E. coli is 50 fold less sensitive to AEC than plant enzymes (IC50 for lysine of 400 μmM−1 mM compared to 10 μM for plants.) (Jacobs et al., 2000)